A detailed comprehension of the role of the thrombospondins (TSPs), including cartilage oligomeric matrix protein (COMP), in the regulation of extracellular matrix (ECM) structure and cellular behavior during tissue genesis and repair is the long-term goal of the proposed studies. Specific focus for the next period of support will be on the following areas. Specific Aim 1. To identify key amino acids for the interactions of COMP with proteins and proteoglycans (PGs). We have found that COMP binds to aggrecan, as well as to integrins in chondrocytes and fibroblasts. In addition, growth plate disorganization is observed in TSP-1-, TSP-3-, and COMP-null mice. We have recently solved the structure of the signature domain of COMP by X-ray crystallography. Based on these data, we plan to determine the molecular basis for the interaction of COMP with collagens, integrins, and glycosaminoglycans (GAGs) by site-directed mutagenesis within the context of the intact molecule and the recombinant signature domain. The aspartic acids that comprise the potential metal ion-dependent adhesion site (MIDAS) and the adjacent to MIDAS motif will be mutated to alanines to establish the importance of these sites in collagen and zinc binding, and matrix assembly. In addition, the specificity of the MIDAS will be explored using peptide libraries. The binding site for GAGs will be identified by X-ray crystallography. Existing structural data, naturally occurring mutations, and published synthetic peptide data will be used to identify amino acids for mutagenesis. This approach will enable us to probe the function of specific motifs within the context of the correctly folded protein. Specific Aim 2. To determine the effects of COMP on cellular phenotype and ECM structure. COMP orchestrates collagen fibril formation, ECM organization, and chondrocyte survival and differentiation during growth plate development. The importance of COMP is underscored by the fact that naturally occurring mutations in it result in skeletal dysplasias. In Aim 1, we will identify the integrin, collagen and GAG binding sites in the COMP molecule. In this aim, we will identify the integrin-mediated cellular responses to COMP in chondrocytes and fibroblasts. We will also explore the effect of the absence of COMP expression using primary cultures of chondrocytes from COMP-null mice. We hypothesize that wild-type COMP promotes cell adhesion, migration, proliferation and survival, while suppressing apoptosis. The specific signaling pathways that mediate the cellular responses to COMP in 2- and 3-D culture systems will be identified. In addition, proliferation, apoptosis and matrix structure in the growth plate of wild-type and COMP-null mice will be analyzed. We will also determine whether or not the integrin and GAG binding sites collaborate with the collagen-binding site to regulate ECM structure. Finally, we will explore the effect of some of the disease causing mutations on the ability of COMP to support cellular processes and matrix assembly. The data from these studies will provide key insights into the function of the signature domain of COMP in terms of its interactions with various proteins, PGs and cells.